Various human disease model animals have been produced using mice or rats. By genetic manipulation, such as introduction of a gene involved in a human disease into a non-human animal, knocking out of a gene homologous to a gene involved in a human disease, or the like, a transgenic non-human animal is produced. The transgenic non-human animal can be used as a human disease model animal. However, it is difficult to estimate the propriety of clinical application to humans based on the results obtained using these animals, as there are anatomical, physiological, and genetic differences between mice or rats and humans. Hence, the use of primates evolutionarily closely related to humans as transgenic animals has been desired.
Production of a transgenic non-human animal requires introduction of a foreign gene(s) such as a gene involved in a human disease into an early embryo of a non-human animal. Conventional methods therefor are DNA microinjection, a method using a virus such as a lentivirus as a vector, and the like. However, microinjection is problematic in that embryonic damage is significant and in lowering gene transfer efficiency (see Hammer, R. E. et al. Nature 315, 680-3 (1985)). It has been necessary to use many oocytes to compensate for such inefficiency. However, it has been difficult to prepare many oocytes in the cases of primates, domestic animals, or the like because of ethical and economical reasons. Accordingly, methods using retroviral vectors have been conducted for livestock (see Chan, A. W. et al., Proc Natl Acad Sci U.S.A. 95, 14028-33 (1998); Hofmann, A. et al. EMBO Rep 4, 1054-60 (2003); and Hofmann, A. et al. Biol Reprod 71, 405-9 (2004)). Such methods using retroviral vectors are problematic in that introduced transgenes are suppressed in the animal body (see Chan, A. W. et al., Science 291, 309-12 (2001)). To remedy such problems of gene suppression, lentiviral vectors have been used for cattle and pigs (see Hofmann, A. et al. EMBO Rep 4, 1054-60 (2003); and Hofmann, A. et al. Biol Reprod 71, 405-9 (2004)).
Regarding primates, production of transgenic rhesus monkeys (Macaca mulatta) has been attempted using retroviruses and lentiviruses (see Chan, A. W. et al., Science 291, 309-12 (2001); and Wolfgang, M. J. et al. Proc Natl Acad Sci U.S.A. 98, 10728-32 (2001)). However, even in the case of using a lentivirus, transgene expression is observed in placenta, but it has never been observed in neonates (see Wolfgang, M. J. et al. Proc Natl Acad Sci U.S.A. 98, 10728-32 (2001)). Production of rhesus monkeys introduced a human Huntington gene has also been reported (see S. H. Yang et al., Nature, vol. 453, No. 7197, 921-??? (2008)), but neither transgene expression in surviving neonates nor germline transmission of the introduced transgene has been observed therein. Rhesus monkeys and crab-eating monkeys (Macaca fascicularis) have been conventionally used as experimental non-human primates. However, these monkeys are problematic in that it is difficult to obtain starter animals for the production of model animals because of the low reproductive rates of these primates, that it takes about 3 years to obtain next-generation animals, and that only about 10 animals per instance can be obtained as next-generation animals.
As described above, high efficiency of gene introduction into embryos has not been achieved with the use of certain mammals such as primates.
With the use of certain animals such as mice, transgenic animals can be produced using ES cells. However, transgenic primate animals wherein a foreign transgene is transmitted to the germline could have not been produced using ES cells. For example, the present inventors have succeeded in production of marmoset ES cells (see International Patent Publication WO2006/029093 Pamphlet), but failed to produce transgenic primates wherein germline transmission takes place via techniques using ES cells.
Meanwhile, a technique referred to as subzonal insemination has been used as an artificial insemination technique (see JP Patent Publication (Kokai) No. H10-185919 A (1998)). This technique accelerates fertilization by injecting spermatozoa into the perivitelline space, which is the space between the vitelline membrane and the zona pellucida. In such cases, oocytes have been treated with a sucrose solution in advance, so as to expand the perivitelline space, for example. This is a technique for artificially causing spermatozoa which have impaired ability for penetrating the clear zone to enter the egg. For artificial insemination, an object is to cause single spermatozoon to enter a single oocyte, but introduction of a large amount of DNA into one cell is not an object. It has not always been possible to achieve a high fertilization rate with this method. Subsequently, this has been replaced by intracytoplasmic sperm injection, which involves direct sperm injection into cytoplasm.